House dust mite allergen, Der f VII, and uses therefor

ABSTRACT

Isolated nucleic acids encoding allergens of the species  Dermatophagoides pteronyssinus  and  Dermatophagoides farinae , Der p VII and Der f VII, respectively, are disclosed. A cDNA encoding a peptide having a Der p VII activity and a predicted molecular weight of about 22, 177 daltons is described. A cDNA encoding a peptide having Der f VII activity is also described. The nucleic acids of the invention can be used as probes to detect the presence of Der p VII or Der f VII nucleic acid in a sample or for the recombinant production of peptides having a Der p VII or Der f VII activity. Peptides having a Der p VII or Der f VII activity can be used in compositions suitable for pharmaceutical administration or methods of diagnosing sensitivity to house dust mite allergens.

RELATED APPLICATIONS

This application claims priority to PCT/AU94/00117 filed on Mar. 11,1994, which in turn is a continuation-in-part of U.S. Ser. No.08/803,141 filed on Mar. 12, 1993 (abandoned) which is a continuation inpart of U.S. Ser. No. 08/081,540 filed on Jun. 22, 1993 (pending).

BACKGROUND OF INVENTION

Approximately 10% of the population become hypersensitized (allergic)upon exposure to antigens from a variety of environmental sources. Thoseantigens that induce immediate and/or delayed types of hypersensitivityare known as allergens (King, T. P., (1976) Adv. Immunol., 23:77-105).These include products of grasses, trees, weeds, animal dander, insects,food, drugs, and chemicals. Genetic predisposition of an individual isbelieved to play a role in the development of immediate allergicresponses (Young, R. P. et al., (1990) Clin. Sci., 79:19) such as atopyand anaphylaxis whose symptoms include hay fever, asthma, and hives.

The antibodies involved in atopic allergy belong primarily to the IgEclass of immunoglobins. IgE binds to basophils, mast cells and dendriticcells via a specific, high-affinity receptor FcεRI (Kinet, J. P., (1990)Curr. Opin. Immunol., 2:499-505). Upon combination of an allergen actingas a ligand with its cognate receptor IgE, FceRI bound to the IgE may becross-linked on the cell surface, resulting in physiologicalmanifestations of the IgE—allergen interaction. These physiologicaleffects include the release of, among other substances, histamine,serotonin, heparin, chemotactic factor(s) for eosinophilic leukocytesand/or leukotrienes C4, D4, and E4, which cause prolonged constrictionof bronchial smooth muscle cells (Hood, L. E. et al., Immunology (2nded.), The Benjamin/Cumming Publishing Co., Inc. (1984)). Hence, theultimate consequence of the interaction of an allergen with IgE isallergic symptoms triggered by the release of the aforementionedmediators. Such symptoms may be systemic or local in nature, dependingon the route of entry of the antigen and the pattern of deposition ofIgE on mast cells or basophils. Local manifestations generally occur onepithelial surfaces at the site of entry of the allergen. Systemiceffects can induce anaphylaxis (anaphylactic shock) which results fromIgE-basophil response to circulating (intravascular) antigen.

Studies with purified allergens have shown that about 80% of patientsallergic to the mite Dermatophagoides pteronyssinus produce IgE reactiveto Der p I and Der p II (Chapman M. D. et al., J. Immunol. (1980)125:587-92; Lind P., J. Allergy Clin. Immunol. (1985) 76:753-61; VanderZee J. S. et al., J. Allergy Clin. Immunol. (1988) 81:884-95). Forabout half of the patients, these specificities constitute 50% of theIgE antimite antibody. The allergen Der p III, recently identified astrypsin, (Stewart G. A. et al., Immunology (1992) 75:29-35) reacts witha similar or higher frequency (Stewart G. A. et al., supra; Ford S. A.et al., Clin. Exp. Allergy (1989) 20:27-31). However, in the onlyquantitative study performed to date, the investigators reported thelevel of IgE binding to be considerably less than Der p I.Electrophoretic techniques (Ford S. A. et al, supra; Bengtsson A. etal., Int. Arch. Allergy Appl. Immunol. (1986) 80:383-90; Lind P. et al.,Scand. J. Immunol. (1983) 17:263-73; Tovey E. R. et al, J. Allergy Clin.Immunol. (1987) 79:93-102) have shown that most sera recognize otherallergens. For example, in the study of Ford et al (supra) Westernblotting showed 8 sera reacting with 1-2 bands, 6 with 3-6 and 3 with agreater number including one with at least 13. In another study, Baldoet al. (Adv. Bioscience (1989) 4:13-31) report the finding of componentsat Mr 30, 26, 25K reacting with 50% of sera. To determine the importanceof particular specificities in the allergic reactions, purifiedallergens would be required for quantitative IgE binding tests and toexamine the frequency and lymphokine profile for T cell reactivity.

Treatment of patients with sensitivity to house dust mites byadministration of increasing doses of house dust extracts has thedrawbacks of potential anaphylaxis during treatment and the possiblenecessity of continuing therapy over a period of several years to buildup sufficient tolerance that results in significant diminution ofclinical symptoms. A therapeutic composition and method of therapy whichavoids these problems would be beneficial.

SUMMARY OF THE INVENTION

This invention provides isolated nucleic acids encoding peptides havingat least one biological activity of Der p VII or Der f VII, proteinallergens of the species Dermatophagoides pteronyssinus andDermatophagoides farinae. Preferred nucleic acids are cDNAs having anucleotide sequence shown in FIGS. 3A and 3B (SEQ ID NO: 1) (Der p VII)and FIGS. 6A and 6B (SEQ ID NO: 6) (Der f VII). The invention alsopertains to peptides encoded by all or a portion of such cDNAs (SEQ IDNO: 1 and SEQ ID NO: 6) and having at least one biological activity ofDer p VII or Der f VII. Also contemplated are isolated nucleic acidswhich hybridize under high stringency conditions (e.g., equivalent to20-27° C. below Tm and 1M NaCl) to a nucleic acid having a nucleotidesequence shown in FIGS. 3A and 3B (SEQ ID NO: 1) or FIGS. 6A and 6B (SEQID NO: 6) or which encodes a peptide comprising all or a portion of anamino acid sequence of FIGS. 3A and 3B (SEQ ID NO: 2)(Der p VII) orFIGS. 6A and 6B (SEQ ID NO: 7)(Der f VII). Nucleic acids which encodepeptides having an activity of Der p VII or Der f VII and having atleast 50% homology with a sequence shown in FIGS. 3A and 3B (SEQ ID NO:2)(Der p VII) or FIGS. 6A and 6B (SEQ ID NO: 7)(Der f VII) are alsofeatured. Peptides having a Der p VII or Der f VII activity produced byrecombinant expression of a nucleic acid of the invention, and peptideshaving a Der p VII or Der f VII activity prepared by chemical synthesisare also featured by this invention. Preferred peptides have the abilityto induce a T cell response, which may include T cell stimulation(measured by, for example, T cell proliferation or cytokine secretion)or T cell nonresponsiveness (i.e., contact with the peptide or a complexof the peptide with an MHC molecule of an antigen presenting cellinduces the T cell to become unresponsive to stimulatory signals orincapable of proliferation). Other preferred peptides, either apart fromor in addition to the ability to induce a T cell response, have theability to bind the dust mite specific IgE of dust mite-allergicsubjects. Such peptides are useful in diagnosing sensitivity to dustmite in a subject. Still other peptides, either apart from or inaddition to the ability to induce a T cell response, have asignificantly reduced ability to bind dust mite-allergic IgE. Suchpeptides are particularly useful as therapeutic agents.

Other preferred peptides comprise an amino acid sequence shown in FIGS.3A and 3B (SEQ ID NO: 2) (Der p VII) or FIGS. 6A and 6B (SEQ ID NO: 7)(Der f VII). In one embodiment, peptides having a Der p VII or Der f VIIactivity and comprising a portion of the amino acid sequence of FIGS. 3Aand 3B (SEQ ID NO: 2) or FIGS. 6A and 6B (SEQ ID NO: 7) are featured.Such peptides are at least about 8-30 amino acids in length, preferablyabout 10-20 amino acids in length, and most preferably about 10-16 aminoacids in length.

Another aspect of the invention features antibodies specificallyreactive with a peptide having a Der p VII or Der f VII activity. Apeptide having an activity of Der p VII or Der f VII can be used incompositions suitable for pharmaceutical administration. Suchcompositions can be used in a manner similar to dust mite extracts totreat or prevent allergic reactions to a dust mite allergen in asubject. Nucleic acids of the invention and peptides having an activityof Der p VII or Der f VII can also be used for diagnosing sensitivity ina subject to a dust mite allergen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the binding frequency of IgE from allergic sera withλgt11-HD6 plaques.

FIG. 2 shows the reactivity of IgE and rabbit anti-house dust miteantibody to purified glutathione-S-transferase fusion product of the HD6insert cloned into pGEX-1.

FIG. 3 is the nucleotide sequence and deduced amino acid sequence of Derp VII clone HD6.

FIG. 4 shows extracts of house dust mites electrophoresed on a 8-18%SDS-PAGE, electroblotted onto nitrocellulose and reacted with pooledallergic serum absorbed with lysates from E coli containing a pGEX-1vector control (lane 1) or pGEX-1 HD6 (lane 2).

FIG. 5 shows the reactivity of affinity purified anti-HD6 antibodies toD. pteronyssinus extracts. Rabbit antibodies were affinity purified onnitrocellulose and used to probe a Western blot of mite extracts,electrophoresed on 8-18% SDS-PAGE and developed with 125_(I-protein) A.

FIG. 6 is the nucleotide sequence and deduced amino acid sequence of Derf VII.

FIGS. 7A, 7B, and 7C is a comparison of the nucleotide sequence anddeduced amino acid sequence of Der f VII and Der p VII. Dots indicate aconsensus in nucleotide sequence between Der f VII and Der p VII.Nucleotide bases which differ between Der f VII and Der p VII areindicated, along with any corresponding amino acid differences.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to isolated nucleic acids encoding peptideshaving at least one biological activity of Der p VII or Der f VII,allergens of the species Dermatophagoides pteronyssinus andDermatophagoides farinae, respectively. Preferably, the nucleic acid isa cDNA comprising a nucleotide sequence shown in FIGS. 3A and 3BA and 3B(SEQ ID NO: 1) (Der p VII) or FIGS. 6A and 6BA and 6B (SEQ ID NO:6) (Derf VII).

The cDNA shown in FIGS. 3A and 3BA and 3B (SEQ ID NO: 1) encodes a Der pVII peptide which includes a 17 amino acid leader sequence encoded bybase 68 through base 118. This leader sequence is not found in themature Der p VII protein, which is encoded by bases 119 through 715. Thededuced amino acid sequence of Der p VII based on this cDNA is alsoshown in FIGS. 3A and 3B (SEQ ID NO: 2). The cDNA encodes a 198 residuemature peptide having a predicted molecular weight of 22,177 Da, nocysteines and a single potential N-linked glycosylation site. A hostcell transfected with an expression vector containing a nucleotidesequence encoding Der p VII was deposited under the Budapest Treaty withthe American Type Culture Collection on Jul. 6, 1993 and assignedaccession number 69.348.

The cDNA shown in FIGS. 6A and 6B (SEQ ID NO: 6) encodes a Der f VIIpeptide. Der f VII peptide is encoded by bases 43 through 681 of thiscDNA sequence. The deduced amino acid sequence of Der f VII based onthis cDNA is shown in FIGS. 6A and 6B (SEQ ID NO: 7). Similar to Der pVII, this Der f VII peptide may contain a leader sequence not found inthe nature protein.

Accordingly, one aspect of this invention pertains to isolated nucleicacids comprising nucleotide sequences encoding Der p VII or Der f VII,fragments thereof encoding peptides having at least one biologicalactivity of Der p VII or Der f VII, and equivalents of such nucleicacids. The term nucleic acid as used herein is intended to include suchfragments and equivalents. The term equivalent is intended to includenucleotide sequences encoding functionally equivalent Der p VII or Der fVII proteins or functionally equivalent peptides having an activity ofDer p VII or Der f VII. As defined herein, a peptide having an activityof Der p VII or Der f VII has at least one biological activity of theDer p VII or Der f VII allergen. Equivalent nucleotide sequences willinclude sequences that differ by one or more nucleotide substitutions,additions or deletions, such as allelic variants, and will also includesequences that differ from the nucleotide sequence encoding Der p VII orDer f VII shown in FIGS. 3A and 3B (SEQ ID NO: 1) or FIGS. 6A and 6B(SEQ ID NO: 6) due to the degeneracy of the genetic code. Equivalentswill also include nucleotide sequences that hybridize under stringentconditions (i.e., equivalent to about 20-27° C. below meltingtemperature (T_(m)) and about 1M salt) to the nucleotide sequence of Derp VII shown in FIGS. 3A and 3B (SEQ ID NO: 1) or Der f VII shown inFIGS. 6A and 6B (SEQ ID NO: 6).

Peptides referred to herein as having an activity of Der p VII or Der fVII or having a Der p VII or Der f VII activity are defined herein aspeptides that have an amino acid sequence substantially corresponding toall or a portion of the amino acid sequence of Der p VII or Der f VIIshown in FIGS. 3A and 3B (SEQ ID NO: 2) or FIGS. 6A and 6B (SEQ ID NO:7), which peptide has at least one biological activity of Der p VII orDer f VII. For example, a peptide having an activity of Der p VII or Derf VII may have the ability to induce a response in Der p VII or Der fVII restricted T cells such as stimulation (e.g., T cell proliferationor cytokine secretion) or to induce T cell non-responsiveness.Alternatively, or additionally, a peptide having an activity of Der pVII or Der f VII may have the ability to bind (to be recognized by)immunoglobulin E (IgE) antibodies of dust mite-allergic subjects.Peptides which bind IgE are useful in methods of detecting allergicsensitivity to Der p VII or Der f VII in a subject. Peptides that do notbind IgE, or bind IgE to a lesser extent than a purified, native Der pVII or Der f VII protein binds IgE are particularly useful astherapeutic agents.

In one embodiment, the nucleic acid is a cDNA encoding a peptide havingan activity of Der p VII or Der f VII. Preferably, the nucleic acid is acDNA molecule comprising at least a portion of the nucleotide sequenceencoding Der p VII shown in FIGS. 3A and 3B (SEQ ID NO: 1) or Der f VIIshown in FIGS. 6A and 6B (SEQ ID NO: 6). A preferred portion of the cDNAmolecules of FIGS. 3A and 3B and FIGS. 6A and 6B includes the codingregion of the molecule.

In another embodiment, the nucleic acid of the invention encodes apeptide having an activity of Der p VII or Der f VII and comprising anamino acid sequence shown in FIG. 3A and 3B (SEQ ID NO:2) (Der p VII) orFIGS. 6A and 6B (SEQ ID NO: 7) (Der f VII). Preferred nucleic acidsencode a peptide having a Der p VII or Der f VII activity and having atleast about 50% homology, more preferably at least about 60% homologyand most preferably at least about 70% homology with the sequence shownin FIGS. 3A and 3B (SEQ ID NO: 1) (Der p VII) or FIGS. 6A and 6B (SEQ IDNO: 6) (Der f VII). Nucleic acids which encode peptides having a Der pVII or Der f VII activity and having at least about 90%, more preferablyat least about 95%, and most preferably at least about 98-99% homologywith a sequence set forth in FIGS. 3A and 3B (SEQ ID NO: 2) (Der p VII)or FIGS. 6A and 6B (SEQ ID NO: 7) (Der f VII) are also within the scopeof the invention. Homology refers to sequence similarity between twopeptides having an activity of Der p VII or Der f VII or between twonucleic acid molecules. Homology can be determined by comparing aposition in each sequence which may be aligned for purposes ofcomparison. When a position in the compared sequence is occupied by thesame base or amino acid, then the molecules are homologous at thatposition. A degree of homology between sequences is a function of thenumber of matching or homologous positions shared by the sequences.

Another aspect of the invention provides a nucleic acid which hybridizesunder high or low stringency conditions to a nucleic acid which encodesa peptide having all or a portion of an amino acid sequence shown inFIGS. 3A and 3B (SEQ ID NO: 2) (Der p VII) or FIGS. 6A and 6B (SEQ IDNO: 7) (Der f VII). Appropriate stringency conditions which promote DNAhybridization, for example, 6.0+ sodium chloride/sodium citrate (SSC) atabout 45° C., followed by a wash of 2.0×SSC at 500 are known to thoseskilled in the art or can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, thesalt concentration in the wash step can be selected from a lowstringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C.

Isolated nucleic acids encoding peptides having an activity of Der p VIIor Der f VII, as described herein, and having a sequence which differsfrom the nucleotide sequences shown in FIGS. 3A and 3B (SEQ ID NO: 1)and FIGS. 6A and 6B (SEQ ID NO: 6) due to degeneracy in the genetic codeare also within the scope of the invention. Such nucleic acids encodefunctionally equivalent peptides (i.e., a peptide having an activity ofDer p VII or Der f VII) but differ in sequence from the sequences ofFIGS. 3A and 3B and FIGS. 6A and 6B due to degeneracy in the geneticcode. For example, a number of amino acids are designated by more thanone triplet. Codons that specify the same amino acid, or synonyms (forexample, CAU and CAC are synonyms for histidine) may result in “silent”mutations which do not affect the amino acid sequence of the Der p VIIor Der f VII protein. However, it is expected that DNA sequencepolymorphisms that do lead to changes in the amino acid sequence of Derp VII or Der f VII will exist within the dust mite population. Oneskilled in the art will appreciate that these variations in one or morenucleotides (up to about 3-4% of the nucleotides) of the nucleic acidsencoding peptides having an activity of Der p VII or Der f VII may existamong individual dust mites due to natural allelic variation. Any andall such nucleotide variations and resulting amino acid polymorphismsare within the scope of this invention. Furthermore, there may be one ormore isoforms or related, cross-reacting family members of Der p VII orDer f VII. Such isoforms or family members are defined as proteinsrelated in function and amino acid sequence to Der p VII or Der f VII,but encoded by genes at different loci.

Fragments of the nucleic acid encoding Der p VII or Der f VII are alsowithin the scope of the invention. As used herein, a fragment of thenucleic acid encoding Der p VII or Der f VII refers to a nucleotidesequence having fewer nucleotides than the nucleotide sequence encodingthe entire amino acid sequence of Der p VII or Der f VII protein andwhich encodes a peptide having an activity of Der p VII or Der f VII(i.e., a peptide having at least one biological activity of the Der pVII or Der f VII allergen) as defined herein.

Preferred nucleic acid fragments encode peptides of at least about 7amino acid residues in length, preferably about 13-40 amino acidresidues in length, and more preferably about 16-30 amino acid residuesin length. Nucleic acid fragments which encode peptides having a Der pVII activity of at least about 30 amino acid residues in length, atleast about 40 amino acid residues in length, at least about 60 aminoacid residues in length, at least about 80 amino acid residues inlength, at least about 100 amino acid residues in length, at least about140 residues in length, and at least about 190 residues in length ormore are also within the scope of this invention. Nucleic acid fragmentswhich encode peptides having a Der f VII activity of at least about 30amino acid residues in length, at least about 40 amino acid residues inlength, at least about 60 amino acid residues in length, at least about80 amino acid residues in length, at least about 100 amino acid residuesin length, at least about 140 residues in length, and at least about 200amino acid residues in length or more are also within the scope of thisinvention. In general, expression of peptides in a transformed host cellis most advantageous where the desired peptide is greater than about 20amino acids in length. Shorter peptides are typically more easilysynthesized chemically.

Nucleic acid fragments within the scope of the invention include thosecapable of hybridizing under high or low stringency conditions withnucleic acids from other animal species for use in screening protocolsto detect Der p VII or Der f VII or allergens that are cross-reactivewith Der p VII or Der f VII. Generally, the nucleic acid encoding apeptide having an activity of Der p VII or Der f VII will be selectedfrom the bases encoding the mature protein, however, in some instancesit may be desirable to select all or part of a peptide from the leadersequence portion of the nucleic acids of the invention. Nucleic acidswithin the scope of the invention may also contain linker sequences,modified restriction endonuclease sites and other sequences useful formolecular cloning, expression or purification of recombinant peptideshaving an activity of Der p VII or Der f VII.

A nucleic acid encoding a peptide having an activity of Der p VII or Derf VII may be obtained from mRNA of the dust mite Dermatophagoidespteronyssinus or Dermatophagoides farinae. It should also be possible toobtain nucleic acids encoding Der p VII or Der f VII fromDermatophagoides pteronyssinus or Dermatophagoides farinae genomic DNA.For example, the gene encoding Der p VII or Der f VII can be cloned fromeither a cDNA or a genomic library in accordance with protocols hereindescribed (see Examples 1 and 2). A cDNA encoding Der p VII or Der f VIIcan be obtained by isolating total mRNA from Dermatophagoidespteronyssinus. Double stranded cDNAs can then be prepared from the totalmRNA. Subsequently, the cDNAs can be inserted into a suitable plasmid orbacteriophage vector using any one of a number of known techniques.Genes encoding Der p VII or Der f VII can also be cloned usingestablished polymerase chain reaction techniques (see Examples 4 and 5)in accordance with the nucleotide sequence information provided by theinvention. The nucleic acids of the invention can be DNA or RNA. Apreferred nucleic acid is a cDNA encoding Der p VII or Der f VII havingthe sequence depicted in FIGS. 3A and 3B (SEQ ID NO: 1) (Der p VII ) orFIGS. 6A and 6B (SEQ ID NO: 6) (Der f VII).

This invention also provides expression vectors containing a nucleicacid encoding a peptide having an activity of Der p VII or Der f VII,operably linked to at least one regulatory sequence. Operably linked isintended to mean that the nucleotide sequence is linked to a regulatorysequence in a manner which allows expression of the nucleotide sequence.Regulatory sequences are art-recognized and are selected to directexpression of the peptide having an activity of Der p VII or Der f VII.Accordingly, the term regulatory sequence includes promoters, enhancersand other expression control elements. Such regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). It should be understoodthat the design of the expression vector may depend on such factors asthe choice of the host cell to be transformed and/or the type of proteindesired to be expressed. In one embodiment, the expression vectorincludes a DNA encoding a peptide having an activity of Der p VII or Derf VII. Such expression vectors can be used to transfect cells to therebyproduce proteins or peptides, including fusion proteins or peptidesencoded by nucleic acids as described herein.

This invention further pertains to a host cell transfected to express apeptide having an activity of Der p VII or Der f VII. The host cell maybe any procaryotic or eucaryotic cell. For example, a peptide having anactivity of Der p VII or Der f VII may be expressed in bacterial cellssuch as E. coli, insect cells (baculovirus), yeast, or mammalian cellssuch as Chinese hamster ovary cells (CHO). Other suitable host cells canbe found in Goeddel, (1990) supra or known to those skilled in the art.

Expression in eucaryotic cells such as mammalian, yeast, or insect cellscan lead to partial or complete glycosylation and/or formation ofrelevant inter- or intra-chain disulfide bonds of recombinant protein.Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari. et al., (1987) Embo J., 6: 229-234), pMFa (Kurjan andHerskowitz, (1982) Cell, 30: 933-943), pJRY88 (Schultz et al., (1987)Gene, 54: 113-123, and pYES2 (Invitrogen Corporation, San Diego,Calif.). Baculovirus vectors available for expression of proteins, incultured insect cells (SF 9 cells) include the pAc series (Smith et al.,(1983) Mol. Cell Biol., 3: 2156-2165) and the pVL series (Lucklow, V.A., and Summers, M. D., (1989) Virology, 170: 31-39). Generally COScells (Gluzman, Y., (1981) Cell, 23: 175-182) are used in conjunctionwith such vectors as pCDM 8 (Aruffo, A. and Seed, B., (1987) Proc. Natl.Acad. Sci. USA, 84: 8573-8577) for transient amplification/expression inmammalian cells, while CHO (dhfr- Chinese Hamster Ovary) cells are usedwith vectors such as pMT2PC (Kaufmnan et al., (1987) EMBO J., 6:187-195) for stable amplification/expression in mammalian cells. VectorDNA can be introduced into mammalian cells via conventional techniquessuch as calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, or electroporation. Suitable methodsfor transforming host cells can be found in Sambrook et al., (MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratorypress (1989)), and other laboratory textbooks.

Expression in procaryotes is most often carried out in E. coli witheither fusion or non-fusion inducible expression vectors. Fusion vectorsusually add a number of NH2 terminal amino acids to the expressed targetgene. These NH2 terminal amino acids often are referred to as a reportergroup. Such reporter groups usually serve two purposes: 1) to increasethe solubility of the target recombinant protein; and 2) to aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the reportergroup and the target recombinant protein to enable separation of thetarget recombinant protein from the reporter group subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne,Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase,maltose E binding protein, or protein A, respectively, to the targetrecombinant protein.

Inducible non-fusion expression vectors include pTrc (Amann et al.,(1988) Gene, 69: 301-315) and pET 11d (Studier et al., Gene ExpressionTechnology: Methods in Enzymology, 185, Academic Press, San Diego,Calif. (1990) 60-89). While target gene expression relies on host RNApolymerase transcription from the hybrid trp-lac fusion promoter inpTrc, expression of target genes inserted into pET 11d relies ontranscription from the T7 gn10-lac 0 fusion promoter mediated bycoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 under the transcriptional control of thelacUV 5 promoter.

One strategy to maximize recombinant Der p VII or Der f VII expressionin E. coli is to express the protein in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,S., Gene Expression Technology: Methods in Enzymology, 185, AcademicPress, San Diego, Calif. (1990) 119-128). Another strategy is be toalter the nucleic acid encoding the Der p VII or Der f VII protein to beinserted into an expression vector so that the individual codons foreach amino acid would be those preferentially utilized in highlyexpressed E. coli proteins (Wada et al., (1992) Nuc. Acids Res., 20:2111-2118). Such alteration of nucleic acids of the invention can becarried out by standard DNA synthesis techniques.

The nucleic acids of the invention can also be chemically synthesizedusing standard techniques. Various methods of chemically synthesizingpolydeoxynucleotides are known, including solid-phase synthesis which,like peptide synthesis, has been fully automated in commerciallyavailable DNA synthesizers (S e.g., Itakura et al., U.S. Pat. No.4,598,049; Caruthers et al., U.S. Pat. No. 4,458,066; and Itakura, U.S.Pat. Nos. 4,401,796 and 4,373,071, incorporated by reference herein).

This invention further pertains to methods of producing peptides thathave an activity of Der p VII or Der f VII. For example, a host celltransfected with a nucleic acid vector directing expression of anucleotide sequence encoding a peptide having an activity of Der p VIIor Der f VII can be cultured under appropriate conditions to allowexpression of the peptide to occur. The peptide may be secreted andisolated from a mixture of cells and medium containing the peptidehaving an activity of Der p VII or Der f VII. Alternatively, the peptidemay be retained cytoplasmically and the cells harvested, lysed and theprotein isolated. A cell culture includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.The peptide having an activity of Der p VII or Der f VII can be isolatedfrom cell culture medium, host cells, or both using techniques known inthe art for purifying proteins including ion-exchange chromatography,gel filtration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for a peptidehaving an activity of Der p VII or Der f VII.

Another aspect of the invention pertains to isolated peptides having anactivity of Der p VII or Der f VII. A peptide having an activity of Derp VII or Der f VII has at least one biological activity of the Der p VIIor Der f VII allergen. For example, a peptide having an activity of Derp VII or Der f VII may have the ability to induce a response in Der pVII or Der f VII specific T cells such as stimulation (T cellproliferation or cytokine secretion) or to induce T cellnon-responsiveness. In one embodiment, a peptide having an activity ofDer p VII or Der f VII stimulates T cells as evidenced by, for example,T cell proliferation or cytokine secretion. In another embodiment,peptides having a Der p VII or Der f VII activity induce T cellnon-responsiveness in which T cells are unresponsive to a subsequentchallenge with a Der p VII or Der f VII peptide following exposure tothe peptide. In yet another embodiment, a peptide, having a Der p VII orDer f VII activity has reduced IgE binding activity compared topurified, native Der p VII or Der f VII protein. A peptide having anactivity of Der p VII or Der f VII may differ in amino acid sequencefrom the Der p VII or Der f VII sequence depicted in FIGS. 3A and 3B(SEQ ID NO:2) (Der p VII) or FIGS. 6A and 6B (SEQ ID NO: 7) (Der f VII)but such differences result in a modified protein which functions in thesame or similar manner as a native Der p VII or Der f VII protein orwhich has the same or similar characteristics of a native Der p VII orDer f VII protein. Various modifications of the Der p VII or Der f VIIprotein to produce these and other functionally equivalent peptides aredescribed in detail herein. The term peptide, as used herein, refers tofull length proteins and polypeptides or peptide fragments thereof.

A peptide can be produced by modification of the amino acid sequence ofthe Der p VII or Der f VII protein shown in FIGS. 3A and 3B (SEQ ID NO:2) (Der p VII) or FIGS. 6A and 6B (SEQ ID NO: 7) (Der f VII), such as asubstitution, addition, or deletion of an amino acid residue which isnot directly involved in the function of the protein. Peptides of theinvention can be at least about 10 amino acid residues in length,preferably about 10-20 amino acid residues in length, and morepreferably about 10-16 amino acid residues in length. Peptides having anactivity of Der p VII or Der f VII and which are at least about 30 aminoacid residues in length, at least about 40 amino acid residues inlength, at least about 60 amino acid residues in length, at least about80 amino acid residues in length, and at least about 100 amino acidresidues in length are also included within the scope of this invention.

Another embodiment of the invention provides a substantially purepreparation of a peptide having an activity of Der p VII or Der f VII.Such a preparation is substantially free of proteins and peptides withwhich the peptide naturally occurs (i.e., other dust mite peptides),either in a cell or when secreted by a cell.

The term isolated as used herein refers to a nucleic acid or peptidethat is substantially free of cellular material or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Such proteins or peptides arealso characterized as being free of all other dust mite proteins.Accordingly, an isolated peptide having an activity of Der p VII or Derf VII is produced recombinantly or synthetically and is substantiallyfree of cellular material and culture medium or substantially free ofchemical precursors or other chemicals and is substantially free of allother dust mite proteins. An isolated nucleic acid is also free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the organism fromwhich the nucleic acid is derived.

Peptides having an activity of Der p VII or Der f VII can be obtained,for example, by screening peptides recombinantly produced from thecorresponding fragment of the nucleic acid of Der p VII or Der f VIIencoding such peptides. In addition, fragments can be chemicallysynthesized using techniques known in the art such as conventionalMerrifield solid phase f-Moc or t-Boc chemistry. For example, the Der pVII or Der f VII protein may be arbitrarily divided into fragments ofdesired length with no overlap of the fragments, or preferably dividedinto overlapping fragments of a desired length. The fragments can beproduced (recombinantly or by chemical synthesis) and tested to identifythose peptides having a Der p VII or Der f VII activity (i.e., theability to induce a T cell response such as stimulation (proliferation,cytokine secretion), nonresponsiveness, and/or has reduced IgE bindingactivity).

In one embodiment, peptides having an activity of Der p VII or Der f VIIcan be identified by the ability of the peptide to stimulate T cells orto induce T cell non-responsiveness. Peptides which stimulate T cells,as determined by, for example, T cell proliferation or cytokinesecretion are defined herein as comprising at least one T cell epitope.T cell epitopes are believed to be involved in initiation andperpetuation of the immune response to the protein allergen which isresponsible for the clinical symptoms of allergy. These T cell epitopesare thought to trigger early events at the level of the T helper cell bybinding to an appropriate HLA molecule on the surface of an antigenpresenting cell, thereby stimulating the T cell subpopulation with therelevant T cell receptor for the epitope. These events lead to T cellproliferation, lymphokine secretion, local inflammatory reactions,recruitment of additional immune cells to the site of antigen/T cellinteraction, and activation of the B cell cascade, leading to theproduction of antibodies. One isotype of these antibodies, IgE, isfundamentally important to the development of allergic symptoms and itsproduction is influenced early in the cascade of events at the level ofthe T helper cell, by the nature of the lymphokines secreted. A T cellepitope is the basic element, or smallest unit of recognition by a Tcell receptor, where the epitope comprises amino acids essential toreceptor recognition. Amino acid sequences which mimic those of the Tcell epitopes and which modify the allergic response to proteinallergens are within the scope of this invention.

Screening peptides for those which retain a Der p VII or Der f VIIactivity as described herein can be accomplished using one or more ofseveral different assays. For example, in vitro, Der p VII or Der f VIIT cell stimulatory activity is assayed by contacting a peptide known orsuspected of having a Der p VII or Der f VII activity with an antigenpresenting cell which presents appropriate MHC molecules in a T cellculture. Presentation of a peptide having a Der p VII or Der f VIIactivity in association with appropriate MHC molecules to T cells inconjunction with the necessary costimulation has the effect oftransmitting a signal to the T cell that induces the production ofincreased levels of cytokines, particularly of interleukin-2 andinterleukin-4. The culture supernatant can be obtained and assayed forinterleukin-2 or other known cytokines. For example, any one of severalconventional assays for interleukin-2 can be employed, such as the assaydescribed in Proc. Natl. Acad. Sci USA, 86: 1333 (1989) the pertinentportions of which are incorporated herein by reference. A kit for anassay for the production of interferon is also available from GenzymeCorporation (Cambridge, Mass.).

Alternatively, a common assay for T cell proliferation entails measuringtritiated thymidine incorporation. The proliferation of T cells can bemeasured in vitro by determining the amount of ³H-labeled thymidineincorporated into the replicating DNA of cultured cells. Therefore, therate of DNA synthesis and, in turn, the rate of cell division can bequantified.

In one embodiment, peptides which have Der p VII or Der f VII T cellstimulating activity (i.e., the peptide comprises at least one T cellepitope) can be identified using an algorithm which predicts thepresence of T cell epitopes in a protein sequence, such as the algorithmdescribed by Hill et al., Journal of Immunology, 147:189-197 (1991). Thealgorithm of Hill et al. predicts the location of T cell epitopes in aprotein by the presence of certain patterns within the sequence whichare likely to bind MHC and therefore may contain T cell epitopes.

In order to determine precise T cell epitopes by, for example, finemapping techniques, a peptide having Der p VII or Der f VII T cellstimulating activity and thus comprising at least one T cell epitope asdetermined by T cell biology techniques is modified by addition ordeletion of amino acid residues at either the amino or carboxy terminusof the peptide and tested to determine a change in T cell reactivity tothe modified peptide. Following this technique, peptides are selectedand produced recombinantly or synthetically. Peptides are selected basedon various factors, including the strength of the T cell response to thepeptide (e.g., stimulation index), the frequency of the T cell responseto the peptide in a population of individuals sensitive to dust miteallergens, and the potential cross-reactivity of the peptide with otherdust mite allergens. The physical and chemical properties of theseselected peptides (e.g., solubility, stability) are examined todetermine whether the peptides are suitable for use in therapeuticcompositions or whether the peptides require modification as describedherein. The ability of the selected peptides or selected modifiedpeptides to stimulate human T cells (e.g., induce proliferation,lymphokine secretion) is then determined as described herein.

In another embodiment, a peptide having a Der p VII or Der f VIIactivity is screened for the ability to induce T cellnon-responsiveness. The ability of a peptide known to stimulate T cells(as determined by one or more of the above described assays), to inhibitor completely block the activity of purified native Der p VII or Der fVII or portion thereof and induce a state of non-responsiveness can bedetermined using subsequent attempts at stimulation of the T cells withantigen presenting cells that present native Der p VII or Der f VII orpeptide having a Der p VII or Der f VII activity following exposure tothe peptide, having a Der p VII or Der f VII activity. If the T cellsare unresponsive to the subsequent activation attempts, as determined byinterleukin-2 synthesis and/or T cell proliferation, a state ofnon-responsiveness has been induced. See, e.g., Gimmi et al., (1993)Proc. Natl. Acad. Sci USA, 90: 6586-6590; and Schwartz (1990) Science,248 1349-1356, for assay systems that can be used as the basis for anassay in accordance with the present invention.

In yet another embodiment, peptides having a Der p VII or Der f VIIactivity are identified by IgE binding activity. For therapeuticpurposes, peptides of the invention preferably do not bind IgE specificfor a dust mite allergen, or bind such IgE to a substantially lesserextent (e.g., at least 100-fold, less, more preferred at least 1000-foldless) than the corresponding purified native dust mite allergen bindssuch IgE. Reduced IgE binding activity refers to IgE binding activitythat is less than that of purified native Der p VII or Der f VIIprotein. If a peptide having a Der p VII or Der f VII activity is to beused as a diagnostic reagent, it is not necessary that the peptide havereduced IgE binding activity compared to the native Der p VII or Der fVII allergen. IgE binding activity of peptides can be determined by, forexample, an enzyme-linked immunosorbent assay (ELISA) using, forexample, sera obtained from a subject, (i.e., an allergic subject) thathas been previously exposed to the native Der p VII or Der f VIIallergen. Briefly, the peptide suspected of having a Der p VII or Der fVII activity is coated onto wells of a microtiter plate. After washingand blocking the wells, antibody solution consisting of the plasma of anallergic subject who has been exposed to a peptide suspected of having aDer p VII or Der f VII activity is incubated in the wells. The plasma isgenerally depleted of IgG before incubation. A labeled secondaryantibody is added to the wells and incubated. The amount of IgE bindingis then quantified and compared to the amount of IgE bound by apurified, native Der p VII or Der f VII protein. Alternatively, the IgEbinding activity of a peptide can be determined by Western blotanalysis. For example, a peptide suspected of having a Der p VII or Derf VII activity is run on a polyacrylamide gel using SDS-PAGE. Thepeptide is then transferred to nitrocellulose and subsequently incubatedwith sera from an allergic subject. After incubation with a labeledsecondary antibody, the amount of IgE bound is then determined andquantified.

Another assay which can be used to determine the IgE binding activity ofa peptide is a competition ELISA assay. Briefly, an IgE antibody pool isgenerated by combining plasma from dust mite allergic subjects that havebeen shown by direct ELISA to have IgE reactive with native Der p VII orDer f VII. This pool is used in ELISA competition assays to compare IgEbinding of native Der p VII or Der f VII and a peptide suspected ofhaving a Der p VII or Der f VII activity. IgE binding for the native Derp VII or Der f VII protein and a peptide suspected of having a Der p VIIor Der f VII activity is determined and quantified.

If a peptide having an activity of Der p VII or Der f VII binds IgE, andis to be used as a therapeutic agent, it is preferable that such bindingdoes not result in the release of mediators (e.g., histamines) from mastcells or basophils. To determine whether a peptide which binds IgEresults in the release of mediators, a histamine release assay can beperformed using standard reagents and protocols obtained, for example,from Amac, Inc. (Westbrook, Me.). Briefly, a buffered solution of apeptide suspected of having a Der p VII or Der f VII activity iscombined with an equal volume of whole heparinized blood from anallergic subject. After mixing and incubation, the cells are pelletedand the supernatants are processed and analyzed using a radioimmunoassayto determine the amount of histamine released.

Peptides having an activity of Der p VII or Der f VII which are to beused as therapeutic agents are preferably tested in mammalian models ofdust mite atopy, such as the mouse model disclosed in Tamura et al.,(1986) Microbiol. Immunol., 30: 883-896, or in U.S. Pat. No. 4,939,239,or in the primate model disclosed in Chiba et al., (1990) Int. Arch.Allergy Immunol., 93: 83-88. Initial screening for IgE binding to apeptide having an activity of Der p VII or Der f VII may be performed byscratch tests or intradermal skin tests on laboratory animals or humanvolunteers, or in in vitro systems such as RAST, RAST inhibition, ELISAassay, RIA (radioimmunoassay), or a histamine release assay, asdescribed above.

It is possible to modify the structure of a peptide having an activityof Der p VII or Der f VII for such purposes as increasing solubility,enhancing therapeutic or prophylactic efficacy, or stability (e.g.,shelf life ex vivo and resistance to proteolytic degradation in vivo).Such modified peptides are considered functional equivalents of peptideshaving an activity of Der p VII or Der f VII as defined herein. Amodified peptide can be produced in which the amino acid sequence hasbeen altered, such as by amino acid substitution, deletion, or addition,to modify immunogenicity and/or reduce allergenicity, or to which acomponent has been added for the same purpose.

For example, a peptide having an activity of Der p VII or Der f VII canbe modified so that it maintains the ability to induce T cellnon-responsiveness and bind MHC proteins without the ability to induce astrong proliferative response or possibly, any proliferative responsewhen administered in immunogenic form. In this instance, criticalbinding residues for T cell receptor function can be determined usingknown techniques (e.g., substitution of each residue and determinationof the presence or absence of T cell reactivity). Those residues shownto be essential to interact with the T cell receptor can be modified byreplacing the essential amino acid with another, preferably similaramino acid residue (a conservative substitution) whose presence is shownto enhance, diminish but not eliminate, or not affect T cell reactivity.In addition, those amino acid residues which are not essential for Tcell receptor interaction can be modified by being replaced by anotheramino acid whose incorporation may enhance, diminish but not eliminate,or not affect T cell reactivity, but does not eliminate binding torelevant MHC.

Additionally, a peptide having an activity of Der p VII or Der f VII canbe modified by replacing an amino acid shown to be essential to interactwith the MHC protein complex with another, preferably similar amino acidresidue (conservative substitution) whose presence is shown to enhance,diminish but not eliminate, or not affect T cell activity. In addition,amino acid residues which are not essential for interaction with the MHCprotein complex but which still bind the MHC protein complex can bemodified by being replaced by another amino acid whose incorporation mayenhance, not affect, or diminish but not eliminate T cell reactivity.Preferred amino acid substitutions for non-essential amino acidsinclude, but are not limited to substitutions with alanine, glutamicacid, or a methyl amino acid.

Another example of modification of a peptide having an activity of Der pVII or Der f VII is substitution of cysteine residues preferably withalanine, serine, threonine, leucine or glutamic acid residues tominimize dimerization via disulfide linkages. In addition, amino acidside chains of fragments of the protein of the invention can bechemically modified. Another modification is cyclization of the peptide.

In order to enhance stability and/or reactivity, a peptide having anactivity of Der p VII or Der f VII can be modified to incorporate one ormore polymorphisms in the amino acid sequence of the protein allergenresulting from any natural allelic variation. Additionally, D-aminoacids, non-natural amino acids, or non-amino acid analogs can besubstituted or added to produce a modified protein within the scope ofthis invention. Furthermore, a peptide having an activity of Der p VIIor Der f VII can be modified using polyethylene glycol (PEG) accordingto the method of A. Sehon and co-workers (Wie et al., supra) to producea protein conjugated with PEG. In addition, PEG can be added duringchemical synthesis of the protein. Other modifications of a peptidehaving an activity of Can f Ior Der f VII include reduction/alkylation(Tarr, Methods of Protein Microcharacterization, J. E. Silver ed.,Humana Press, Clifton N.J. 155-194 (1986)); acylation (Tarr, supra);chemical coupling to an appropriate carrier (Mishell and Shiigi, eds,Selected Methods in Cellular Immunology, W H Freeman, San Francisco,Calif. (1980), U.S. Pat. No. 4,939,239; or mild formalin treatment(Marsh, (1971) Int. Arch. of Allergy and Appl. Immunol., 41: 199-215).

To facilitate purification and potentially increase solubility of apeptide having an activity of Der p VII or Der f VII, it is possible toadd an amino acid fusion moiety to the peptide backbone. For example,hexa-histidine can be added to the protein for purification byimmobilized metal ion affinity chromatography (Hochuli, E. et al.,(1988) Bio/Technology, 6: 1321-1325). In addition, to facilitateisolation of peptides free of irrelevant sequences, specificendoprotease cleavage sites can be introduced between the sequences ofthe fusion moiety and the peptide. In order to successfully desensitizea subject to Der p VII or Der f VII protein or related allergen, it maybe necessary to increase the solubility of the protein by addingfunctional groups to the protein, or by omitting hydrophobic regions ofthe protein. Functional groups, such as charged amino acids and chargedamino acid pairs are suitable for increasing solubility when added tothe amino or carboxy terminus of the peptide.

To potentially aid proper antigen processing of T cell epitopes withinDer p VII or Der f VII, canonical protease sensitive sites can beengineered between regions, each comprising at least one T cell epitopevia recombinant or synthetic methods. For example, charged amino acidpairs, such as KK or RR, can be introduced between regions within aprotein or fragment during recombinant construction thereof. Theresulting peptide can be rendered sensitive to cleavage by cathepsinand/or other trypsin-like enzymes which would generate portions of theprotein containing one or more T cell epitopes. In addition, suchcharged amino acid residues can result in an increase in the solubilityof the peptide.

Site-directed mutagenesis of a nucleic acid encoding a peptide having anactivity of Der p VII or Der f VII can be used to modify the structureof the peptide by methods known in the art. Such methods may, amongothers, include polymerase chain reaction (PCR) with oligonucleotideprimers bearing one or more mutations (Ho et al., (1989) Gene, 77:51-59) or total synthesis of mutated genes (Hostomsky, Z. et al., (1989)Biochem. Biophys. Res. Comm, 161: 1056-1063). To enhance recombinantprotein expression, the aforementioned methods can be applied to changethe codons present in the cDNA sequence of the invention to thosepreferentially utilized by the host cell in which the recombinantprotein is being expressed (Wada et al., supra).

Another aspect of the invention pertains to an antibody specificallyreactive with a peptide having an activity of Der p VII or Der f VII.The antibodies of this invention can be used to standardize allergenextracts or to isolate the naturally-occurring or native form of Der pVII or Der f VII. For example, by using peptides having an activity ofDer p VII or Der f VII based on the cDNA sequence of Der p VII or Der fVII, anti-protein/anti-peptide antisera or monoclonal antibodies can bemade using standard methods. A mammal such as a mouse, a hamster orrabbit can be immunized with an immunogenic form of the peptide (e.g.,Der p VII or Der f VII protein or an antigenic fragment which is capableof eliciting an antibody response). Techniques for conferringimmunogenicity on a protein or peptide include conjugation to carriersor other techniques well known in the art. A peptide having an activityof Der p VII or Der f VII can be administered in the presence ofadjuvant. The progress of immunization can be monitored by detection ofantibody titers in plasma or serum. Standard ELISA or other immunoassaycan be used with the immunogen as antigen to assess the levels ofantibodies.

Following immunization, anti-Der p VII or anti-Der f VII antisera can beobtained and, if desired, polyclonal anti-Der p VII or anti-Der f VIIantibodies isolated from the serum. To produce monoclonal antibodies,antibody producing cells (lymphocytes) can be harvested from animmunized animal and fused by standard somatic cell fusion procedureswith immortalizing cells such as myeloma cells to yield hybridoma cells.Such techniques are well known in the art, for example the hybridomatechnique originally developed by Kohler and Milstein, (1975) Nature,495-497) as well as other techniques such as the human B cell hybridomatechnique (Kozbar et al., (1983) Immunology Today, 4: 72) and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a peptide having anactivity of Der p VII or Der f VII and the monoclonal antibodiesisolated.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with the peptide having anactivity of Dr p VII or Der f VII. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. For example,F(ab′)₂ fragments can be generated by treating antibody with pepsin. Theresulting F(ab′)₂ fragment can be treated to reduce disulfide bridges toproduce Fab′ fragments. The antibody of the present invention is furtherintended to include bispecific and chimeric molecules having an anti-Derp VII or anti-Der f VII portion.

Another aspect of this invention provides T cell clones and soluble Tcell receptors specifically reactive with a peptide having an activityof Der p VII or Der f VII. Monoclonal T cell populations (i.e., T cellsgenetically identical to one another and expressing identical T cellreceptors) can be derived from an individual sensitive to Der p VII orDer f VII, followed by repetitive in vitro stimulation with a Der p VIIor Der f VII protein or peptide having an activity of Der p VII or Der fVII in the presence of MHC-matched antigen-presenting cells. Single Derp VII or Der f VII MHC responsive cells can then be cloned by limitingdilution and permanent lines expanded and maintained by periodic invitro restimulation. Alternatively, Der p VII or Der f VII specific T-Thybridomas can be produced by a technique similar to B cell hybridomaproduction. For example, a mammal, such as a mouse, is immunized with apeptide having an activity of Der p VII or Der f VII, T cells are thenpurified and fused with an autonomously growing T cell tumor line. Fromthe resulting hybridomas, cells responding to a peptide having anactivity of Der p VII or Der f VII are selected and cloned. Proceduresfrom propagating monoclonal T cell populations are described in Cellularand Molecular Immunology (Abul K. Abbas et al. ed.), W. B. SaundersCompany, Philadelphia, Pa. (1991) page 139. Soluble T cell receptorsspecifically reactive with a peptide having an activity of Der p VII orDer f VII can be obtained by immunoprecipitation using an antibodyagainst the T cell receptor as described in Immunology: A Synthesis(Second Edition), Edward S. Golub et al., ed., Sinauer Associates, Inc.,Sunderland, Mass. (1991) pages 366-269.

T cell clones specifically reactive with a peptide having an activity ofDer p VII or Der f VII can be used to isolate and molecularly clone thegene encoding the relevant T cell receptor. In addition, a soluble Tcell receptor specifically reactive with a peptide having an activity ofDer p VII or Der f VII can be used to interfere with or inhibitantigen-dependent activation of the relevant T cell subpopulation, forexample, by administration to an individual sensitive to Der p VII orDer f VII. Antibodies specifically reactive with such a T cell receptorcan be produced according to the techniques described herein. Suchantibodies can be used to block or interfere with the T cell interactionwith peptides presented by MHC.

Exposure of allergic subjects to peptides having an activity of Der pVII or Der f VII and which have T cell stimulating activity, may causethe appropriate T cell subpopulations to become non-responsive to therespective protein allergen (e.g., fail to stimulate an immune responseupon such exposure). In addition, such administration may modify thelymphokine secretion profile as compared with exposure to thenaturally-occurring protein allergen or portion thereof (e.g., result ina decrease of IL-4 and/or an increase in IL-2). Furthermore, exposure topeptides having an activity of Der p VII or Der f VII which have T cellstimulating activity may influence T cell subpopulations which normallyparticipate in the response to the allergen such that these T cells aredrawn away from the site(s) of normal exposure to the allergen (e.g.,nasal mucosa, skin, and lung) towards the site(s) of therapeuticadministration of the protein or fragment derived therefrom. Thisredistribution of T cell subpopulations may ameliorate or reduce theability of an individual's immune system to stimulate the usual immuneresponse at the site of normal exposure to the allergen, resulting in adiminution in allergic symptoms.

A peptide having an activity of Der p VII or Der f VII when administeredto a subject sensitive to dust mite allergens is capable of modifyingthe B cell response, T cell response, or both the B cell and the T cellresponse of the subject to the allergen. As used herein, modification ofthe allergic response of a subject to a dust mite allergen can bedefined as non-responsiveness or diminution in symptoms to the allergen,as determined by standard clinical procedures (See e.g., Varney et al.,(1990) British Medical Journal, 302: 265-269), including diminution indust mite induced asthmatic symptoms. As referred to herein, adiminution in symptoms includes any reduction in the allergic responseof a subject to the allergen following a treatment regimen with apeptide of the invention. This diminution in symptoms may be determinedsubjectively (e.g., the patient feels more comfortable upon exposure tothe allergen), or clinically, such as with a standard skin test.

Peptides or antibodies of the present invention can also be used fordetecting and diagnosing sensitivity to Der p VII or Der f VII. Forexample, this can be done in vitro by combining blood or blood productsobtained from a subject to be assessed for sensitivity with peptidehaving an activity of Der p VII or Der f VII, under conditionsappropriate for binding of components in the blood (e.g., antibodies, Tcells, B cells) with the peptide(s) and determining the extent to whichsuch binding occurs. Other diagnostic methods for allergic diseaseswhich the peptides or antibodies of the present invention can be usedinclude radio-allergosorbent test (RAST), paper radioimmunosorbent test(PRIST), enzyme linked immunosorbent assay (ELISA), radioimmunoassays(RIA), immuno-radiometric assays (IRMA), luminescence immunoassays(LIA), histamine release assays and IgE immunoblots.

The present invention further provides methods of detecting and treatingsensitivity in a subject to Der p VII or Der f VII. The presence insubjects of IgE specific for Der p VII or Der f VII and the ability of Tcells of the subjects to respond to T cell epitopes of Der p VII or Derf VII can be determined by administering to the subject an ImmediateType Hypersensitivity test and/or a Delayed Type. Hypersensitivity test(See e.g., Immunology (1985) Roitt, I. M., Brostoff, J., Male, D. K.(eds), C. V. Mosby Co., Gower Medical Publishing, London, N.Y., pp.19.2-19.18; pp.22.1-22.10) utilizing a peptide having an activity of Derp VII or Der f VII, or a modified form of a peptide having an activityof Der p VII or Der f VII, each of which binds IgE specific for theallergen. The same subjects are administered a Delayed TypeHypersensitivity test prior to, simultaneously with, or subsequent toadministration of the Immediate Type Hypersensitivity test. Of course,if the Immediate Type Hypersensitivity test is administered prior to theDelayed Type Hypersensitivity test, the Delayed Type Hypersensitivitytest would be given to those subjects exhibiting a specific ImmediateType Hypersensitivity reaction. The Delayed Type Hypersensitivity testutilizes a peptide having an activity of Der p VII or Der f VII whichhas human T cell stimulating activity and which does not bind IgEspecific for the allergen in a substantial percentage of the populationof subjects sensitive to the allergen (e.g., at least about 75%). Thosesubjects found to have both a specific Immediate type Hypersensitivityreaction and a specific Delayed Type Hypersensitivity reaction areadministered an amount of a composition suitable for pharmaceuticaladministration. The composition comprises the peptide having an activityof Der p VII or Der f VII as used in the Delayed Type Hypersensitivitytest and a pharmaceutically acceptable carrier or diluent.

A peptide having an activity of Der p VII or Der f VII can be used inmethods of diagnosing, treating, or preventing allergic reactions to adust mite allergen or a cross-reactive protein allergen. Thus, thepresent invention provides compositions suitable for in vitro use andpharmaceutical administration comprising an amount of at least onepeptide having an activity of Der p VII or Der f VII. Pharmaceuticalcompositions typically will be formulated with a pharmaceuticallyacceptable carrier.

Where a composition according to the invention is intended foradministration to a subject to be desensitized, such administration canbe carried out using known procedures, at dosages and for periods oftime effective to reduce sensitivity (i.e., reduce the allergicresponse) of the subject to a dust mite allergen. The term subject isintended to include living organisms in which an immune response can beelicited, e.g., mammals. Examples of subjects include humans, dogs,cats, mice, rats, and transgenic species thereof. An amount of at leastone peptide having an activity of Der p VII or Der f VII necessary toachieve a therapeutic effect may vary according to factors such as thedegree of sensitivity of the subject to dust mite, the age, sex, andweight of the subject, and the ability of a peptide having an activityof Der p VII or Der f VII to elicit an antigenic response in thesubject. Dosage regima may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The active compound (i.e., a peptide having an activity of Der p VII orDer f VII) may be administered in a convenient manner such as byinjection (subcutaneous, intravenous, etc.), oral administration,inhalation, transdermal application, or rectal administration. Dependingon the route of administration, the active compound may be coated in amaterial to protect the compound from the action of enzymes, acids andother natural conditions which may inactivate the compound.

To administer a peptide having an activity of Der p VII or Der f VII byother than parenteral administration, it may be necessary to coat thepeptide with, or co-administer the peptide with, a material to preventits inactivation. For example, a peptide having an activity of Der p VIIor Der f VII may be administered to an individual in an appropriatecarrier, diluent or adjuvant, co-administered with enzyme inhibitors orin an appropriate carrier such as liposomes. Pharmaceutically acceptablediluents include saline and aqueous buffer solutions. Adjuvant is usedin its broadest sense and includes any immune stimulating compound suchas interferon. Adjuvants contemplated herein include resorcinols,non-ionic surfactants such as polyoxyethylene oleyl ether andn-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatictrypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.Liposomes include water-in-oil-in-water CGF emulsions as well asconventional liposomes (Strejan et al., (1984) J. Neuroimmunol., 2: 27).For purposes of inducing T cell nonresponsiveness, the composition ispreferably administered in non-immunogenic form, e.g., one that does notcontain adjuvant.

The active compound may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating activecompound (i.e., a peptide having an activity of Der p VII or Der f VII)in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient (i.e., at least one peptide having an activity of Der p VIIor Der f VII) plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

When the peptide having an activity of Der p VII or Der f VII issuitably protected, as described above, the peptide may be orallyadministered, for example, with an inert diluent or an assimilableedible carrier. The peptide and other ingredients may also be enclosedin a hard or soft shell gelatin capsule, compressed into tablets, orincorporated directly into the individual's diet. For oral therapeuticadministration, the active compound may be incorporated with excipientsand used in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 5 to about 80% of theweight of the unit. The amount of active compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

As used herein “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the therapeuticcompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in subjects.

The present invention also provides a composition comprising at leasttwo peptides having an activity of Der p VII or Der f VII (e.g., aphysical mixture of at least two peptides), each having T cellstimulating activity. For example, at least two peptides each having asactivity of Der p VII can be combined or at least two peptides eachhaving an activity of Der f VII can be combined, or at least one peptidehaving an activity of Der p VII and at least one peptide having anactivity of Der f VII can be combined and administered. Alternatively, apeptide having at least two regions, each having T cell stimulatingactivity (i.e., each region comprising at least one T cell epitope) canbe administered to an allergic subject. Such a peptide can have at leasttwo regions derived from the same allergen, Der p VII or Der f VII, or acombination of Der p VII and Der f VII. A composition of two peptides ora peptide having at least two regions can be administered to a subjectin the form of a composition with a pharmaceutically acceptable carrieras hereinbefore described. An amount of one or more of such compositionscan be administered simultaneously or sequentially to a subjectsensitive to a dust mite allergen to treat such sensitivity. Suchcompositions may be useful for the manufacture of a medicament fortreating sensitivity to house dust mites in an individual.

The cDNA (or the mRNA which served as a template during reversetranscription) encoding a peptide having an activity of Der p VII or Derf VII can be used to identify similar nucleic acid sequences in anyvariety or type of animal and, thus, to molecularly clone genes whichhave sufficient sequence homology to hybridize to the cDNA encoding apeptide having an activity of Der p VII or Der f VII. Thus, the presentinvention includes not only peptides having an activity of Der p VII orDer f VII, but also other proteins which may be allergens encoded by DNAwhich hybridizes to DNA of the present invention.

Isolated peptides that are immunologically related to Der p VII or Der fVII, such as by antibody cross-reactivity or T cell cross-reactivity,other than those already identified, are within the scope of theinvention. Such peptides bind antibodies specific for the protein andpeptides of the invention, or stimulate T cells specific for the proteinand peptides of this invention.

A peptide having an activity of Der p VII or Der f VII (i.e., Der p VIIor Der f VII produced recombinantly or by chemical synthesis) is free ofall other dust mite proteins and, thus, is useful in the standardizationof allergen extracts which are key reagents for the diagnosis andtreatment of dust mite hypersensitivity. In addition, such a peptide isof a consistent, well-defined composition and biological activity foruse in preparations which can be administered for therapeutic purposes(e.g., to modify the allergic response of a subject sensitive to dustmite). Such peptides can also be used to study the mechanism ofimmunotherapy of Dermatophagoides pteronyssinus and Dermatophagoidesfarinae allergy and to design modified derivatives or analogs useful inimmunotherapy.

Work by others has shown that high doses of allergen extracts generallyproduce the best results during immunotherapy (i.e., best symptomrelief). However, many subjects are unable to tolerate large doses ofsuch extracts due to systemic reactions elicited by the allergens andother components within these preparations. A peptide having an activityof Der p VII or Der f VII according to the invention has the advantageof being free of all other dust mite proteins, and thus are safer andmore suitable for therapeutic uses.

It is now also possible to design an agent or a drug capable of blockingor inhibiting the ability of a dust mite allergen to induce an allergicreaction in sensitive subjects. Such agents could be designed, forexample, in such a manner that they would bind to relevant anti-Der pVII or anti-Der f VII IgE molecules, thus preventing IgE-allergenbinding, and subsequent mast cell/basophil degranulation. Alternatively,such agents could bind to cellular components of the immune system,resulting in suppression or desensitization of the allergic responses todust mite allergens. A non-restrictive example of this is the use ofpeptides including B or T cell epitopes of Der p VII or Der f VII, ormodifications thereof, based on the cDNA protein structure of Der p VIIor Der f VII to suppress the allergic response to a dust mite allergen.This could be carried out by defining the structures of fragmentsencoding B and T cell epitopes which affect B and T cell function in invitro studies with blood components from subjects sensitive to dustmite.

The invention is further illustrated by the following examples whichshould not be construed as further limiting the subject invention. Thecontents of all references and published patent applications citedthroughout this application are hereby incorporated by reference.

EXAMPLE 1 Isolation of Clone HD6 from a λgt11 cDNA Library

A λgt11 cDNA library was prepared from live adult Dermatophagoidespteronyssinus purchased from the Commonwealth Serum Laboratories,Parkville, Australia (Thomas, W., et al., Int Arch Allergy Appl Immunol(1988) 85:127-9). The library was prepared according to Chua et al. (J.Exp. Med. (1988) 167:175-182) based upon the method of Young and Davis(Proc. Natl. Acad. Sci. USA (1983) 80:1194-1198) and Gubler and Hoffmnan(Gene (1983) 2:263-299). Polyadenylated mRNA was isolated from a D.pteronyssinus culture and cDNA synthesized by the RNaseH method (Gublerand Hoffmnan, supra) using a kit (Amersham International, Bucks). Afterthe addition of EcoRI linkers the cDNA was ligated into λgt11 and platedin E. coli Y1090 (r−) (Promega Biotec, Madison, Wis.) to produce alibrary of 5×10⁵ recombinants.

Allergic serum was used to probe the λgt11 library. IgE plaqueimmunoassays were conducted by a standard procedure (Chua, K. Y., etal., Int Arch Allergy Appl Immunol (1990) 91:118-23) using 20 000 pfu on14.5 cm petri dishes. Briefly, an overnight culture of E. coli Y1090(Huynh, T. V. et al. Constructing and Screening cDNA Libraries in gt10and gt11 in: A Practical Approach, Oxford IRL Press, 1986, pp 48-78) wasdiluted 1/50 in L broth and incubated at 37° C. to an OD₆₅₀ of 0.6. Thebacteria were pelleted and resuspended in 400 μl for every 50 ml ofbroth. For 14.5-cm Petri dishes, 300 μl of Y1090 were incubated with 10⁴pfu phage for 30 minutes at room temperature and then plated on LB agarin 9 ml of 0.7% agar overlay and incubated for 3 hours at 42° C. (whenplaques usually become visible). At this time a nitrocellulose filter,which had been saturated with 10 mM isopropyl β-D-thiogalactoside anddried, was placed on top of the lawn. The incubation continued overnightat 37° C. The filter was then removed and washed in 0.01 MTris-hydrochloride, 0.15 M NaCl, 0.05% Tween 20 v/v, pH 8, (TNT) bufferwith gentle rocking for 20 minutes. The filter was then incubated withsera from mite allergic children for 2 hours at room temperature withrocking and then washed three times for 30 minute periods with TNT. Thesera used was first diluted 50:50 with an E. coli extract (Huynh et al.,supra), incubated overnight then clarified by centrifugation (3,000 g 10minutes). Non-fat milk and sodium azide were added to 5 and 0.02%,respectively. To develop the IgE reactivity the filter was rocked in asolution of ¹²⁵I-labelled anti-IgE for 2 hours at room temperaturefollowed by three 30-minute washes with TNT. The anti-IgE was a mousemonoclonal 2.1.5 (available from Silenus Laboratories Pty. Ltd.,Hawthorn, Victoria) and was used at 30 ng/ml coupled with 10⁵ dpm/ng¹²⁵I in TNT (Stewart, G. A., et al. Int. Arch. Allergy Appl. Immunol.(1988) 86:9-18). It was labelled by the chloramine T method. The filterwas autoradiographed with intensifying screens, usually for 48 hours at−70° C.

A λgt11 derived clone HD6 from the D. pteronyssinus cDNA library wasplaque purified (see Maniati et al. Molecular Cloning: A LaboratoryManual, (1982) Cold Spring Harbor) because it showed high IgE bindingactivity to a mite allergic serum (obtained from a child attending theallergy clinic at the Taiwan University Hospital, Taipei, R.O.C.) by theplaque radioimmune assay described above. To determine the number ofsera with IgE binding for this clone, the λgt11-HD6 was plated at 1 000pfu on a 90 cm petri dish and a nitrocellulose lift prepared for animmunoassay as outlined in Young and Davis (1983) supra, withmodifications as detailed in Chua et al. Int. Arch. Allergy Appl.Immunol. (1990) 91:118-123. The filter was cut into segments and IgEimmunoassays performed with 20 individual sera obtained from the RoyalChildren's Hospital Melbourne (Dr. D. Hill) (FIG. 1). Strong reactivitywas found with 6 sera and in another series with 8/18. A hyper IgE serumtested at 1 000 IU/ml did not show binding, nor did a serum from a childallergic to only rye grass (see bottom two segments in right-hand columnof FIG. 1).

To estimate the size of the IgE binding molecule encoded by the phage,DNA from purified clones was isolated by a polyethyleneglycolprecipitation procedure (Chua, K. Y. et al. J. Exp. Med. (1988)167:175-182) and the 812 bp DNA insert found in the λgt11-HD6 wasreleased by EcoRI digestion (Toyobo, Osaka, Japan) and subcloned intothe same site in the glutathione-S-transferase fusion vector pGEX-1(Smith, D. B. et al., Gene (1988) 67:31-40) and used to transform E.coli TG-1. The protein expressed by this construct was isolated fromcrude bacterial lysates under non-denaturing conditions by affinitychromatography on immobilized a glutathione (as described in Smith etal. Gene (1988) 67:31-40). The fusion protein was then examined byWestern blotting. For Western Blot Analysis, proteins were transferredto nitrocellulose (Bio-Rad transblot) by the protocol of Burnette(Burnette, W. N., Anal Biochem (1981) 112:195-203) and immunoassays wereperformed as for the plaque radioimmune assays with allergic sera and¹²⁵j-anti IgE or with rabbit antibodies and ¹²⁵I-protein A as describedin Greene, W. K., et al., Int Arch Allergy Appl Immunol (1990) 92:30-8.

Expression in pGEX-1 resulted in a protein(s) which migrated as adoublet with a Mr of 53-55 K and reacted by Western blotting with rabbitanti-house dust mite serum (FIG. 2, lane 1). Two allergic sera reactedwith this doublet (FIG. 2, lanes 3 and 5) but not to a hyper IgE serumat 1000 IU/ml (FIG. 2, lane 4) or normal rabbit serum (FIG. 2, lane 2).The IgE binding protein, allowing for the contribution of the 27 Kglutathione transferase would therefore be about Mr 27. This, as will bedescribed below, contains residues from the leader sequence and thosefrom the 5′ untranslated region.

EXAMPLE 2 DNA Sequence Analysis of Clone HD6

The 812 bp insert of clone HD6 was cloned into the M13 vectors mp18 andmp19 (see Messing Methods Enzymology (1983) 101:20) for sequencingperformed in both directions using −40, universal and internal primers(Messing, supra). Dideoxynucleotide sequencing (Sambrook, J., et al.Molecular Cloning. A Laboratory Manual. 2nd Edition. Cold Spring Harbor:Cold Spring Harbor Laboratory Press, 1989) was performed using aSequenase 2.0 kit (IBI, New Haven, USA) with ³²P-dATP and a BioradSequi-gen electrophoresis apparatus. Following sequence analysis withthe universal and internal primers, three primers based on the cDNAsequence of Der p VII were produced and sequencing conducted. Thesequences of the primers are as follows: (1) GATCCAATTCACTATGAT,(bases119-136 in FIG. 3, SEQ ID NO:3); (2) GGTGAATTAGACATGCG (bases 272-288 inFIGS. 3, SEQ ID NO:4); and (3) TCAATTTTGGATCCAATTTTCGCT (bases 584-607,SEQ ID NO:5).

The DNA insert was found to have 812 bases with an open reading framebeginning at the 5′ end, consistent with its expression as a fusion fromλgt11 and pGEX-1, and ending at a stop codon TAG (713-715) (FIG. 3). Thesequence of the translated protein appeared to begin at the adjacentinitiation ATGs at nucleotides 68-70 and 71-73. This is followed bynucleotides encoding a typical, predominantly hydrophobic, leadersequence (Von Hiejne, G. J. Mol. Biol. (1985) 184:99-105) predicted tobe 17 residues long, and then a sequence encoding a further 198 residuesending at the TAG codon at nucleotides 713-715. This reading frame wasconfirmed by using PCR (as described in Saiki et al. Science (1988)239:487-491) to clone DNA encoding an antigenic product of the correctMr in pGEX starting at the predicted N-terminal Asp encoded bynucleotide 119-121. The fusion protein from this construct was producedat far higher yields than the fusion which contained the leader peptide(pGEX-1). The 3′ untranslated region contained a polyadenylation signalAATAAA at 765-770 (underlined in FIGS. 3) and a polyA tail. A potentialN-glycosylation site, Asn Ala Thr, is encoded by nucleotides 518-526(see FIGS. 3 underlined). No homology was found to sequences in theGenpept 71.0, EMBL 30.0 and Swiss-Prot 21 databases. The predictedmolecular weight of the translated polypeptide was 23,865 daltons and22,177 daltons without the leader sequence.

EXAMPLE 3 Nature of the Allergen Der p VII in Mite Extracts

As a first step to identifying the Der p VII native protein allergen, apool of allergic serum obtained as described previously was absorbedwith an equal volume of pGEX-1 HD6 lysate or a control vector lysate(Greene and Thomas Molec. Immunol. (1992) 29:259-262). The serum wasthen used for IgE Western blotting of house dust mite extracts separatedby SDS-PAGE performed according to Laemmli (Laemmli, U.K., Nature (1970)227:680-5) with an 8-18% gradient in 10-12 cm gel assemblies or 13% miniprotean II apparatus (Bio-Rad, Richmond, Va., USA). For dust miteextracts, samples were loaded at 0.1 mg protein/track. For bacteria,cultures were centrifuged and the pellets suspended at 0.01 of theculture volume and 10 μl added to sample buffer for electrophoresis.Purified proteins were electrophoresed at 2-5 μl/track. Compared to theserum absorbed with vector control (FIG. 4, lane 2), the HD6 fusionprotein absorbed serum (FIG. 4, lane 1) showed a loss of reactivity tobands with Mr of 29, 27 and 11.5 K.

To examine this further, rabbit antibodies to the HD6 protein wereaffinity purified from a hyperimmune serum using nitrocellulose filterslifted from plates confluent with λgt11-HD6 plaques. Briefly, antibodieswith specificities for the allergen expressed by the λgt11 clones wereisolated from a hyperimmune rabbit anti-D. pteronyssinus serum (Greene,W. K., et al., Int Arch Allergy Appl Immunol (1990) 92:30-8) (producedby repeated injections into a rabbit of mite extract) by affinitypurification using a nitrocellulose filter blotted on a plaque lawn(Ozaki, L. S., et al., J. Immunol Methods (1986) 89:213-9) as theabsorbant. λgt11 derived phage (clone HD6) were plated at 10 000 pfu per90 cm petri dishes and overlaid with nitrocellulose saturated withisopropyl-β-D-thiogalactopyranoside (IPTG) under the same conditionsused to screen the library. After overnight incubation, the filter wasflipped to expose the other side to the lawn and incubated for 2 hoursat 37° C. The filter was then washed in the TNT buffer, (0.01 M Trishydrochloride, 0.15 NaCl, 0.05% Tween 20 v/v pH 8.0). One ml of rabbitantiserum which had been incubated overnight in 1 ml of a lysate ofλgt11 lysogen was diluted to 20 mls with TNT and skim milk powder addedto 5%. Aliquots of 5 ml were then rocked in petri dishes containing thefilters for 1 hour at room temperature. The filters were washed threetimes in TNT and incubated for 15 minutes at room temperature in 0.1 Mglycine, 0.15 M NaCl pH2.6 to elute the antibodies. Each 5 ml eluate wasthen neutralized by adding 650 μl of 100 mM Tris and 1.5 m NaCl, 50 ml1% sodium azide and 0.25 g skim milk. The solution was dialysed againstPBS.

The affinity purified antibody was then absorbed with E. coli lysateused to develop Western blots as described, on the house dust miteextract and found to react with bands of Mr 29, 27 and 24 (FIG. 5). Thespecificity of the reactivity was further checked by absorbing theaffinity purified antibodies with a pGEX-HD6 lysate expressing theprotein (FIG. 5, lane 3) or a control pGEX construct, pQEX-D15 (FIG. 5,lane 2). The serum absorbed with HD6 (lane 3) lost reactivity to allbands. The affinity purification therefore shows that antibodies to theallergen have specificities for components at Mr 29, 27 and 24 K. Thesame pattern of multiple binding to that described above with extractsprepared from CSL mites was also found with another extract fromHollister-Stier Laboratories, Spokane, Wash., USA.

The finding that antibodies to the HD6 lysate reacted specifically to atleast 3 bands on Western blotting has implications for determining thenumber of allergens recognized by individual mite allergic patients. Themultiple bands were found for the two independent extracts examined andthe absorption studies with allergic serum showed that the 29 and 27 Kbands had IgE reactivity and that this recombinant molecule appeared toabsorb out all of the reactivity to each band. It is not, however, knownfrom this investigation if all patients react with each band. Becausethe Western analysis was performed using reducing conditions and thebands had Mr greater than that calculated from the translated sequence,the different forms of the allergens may be interpreted as differentglycosylation products. This can be confirmed with some caution taken tocontrol for denaturation by the deglycosylation procedures. The patternnevertheless indicates that the number of allergic specificities is lessthan that indicated by electrophoretic procedures, a significantobservation for immunotherapeutic strategies using purified, recombinantor peptide allergens. Alternatively, the different Mr bands reactingwith the anti-HD6 antibody may indicate the presence of related or crossreactive allergens.

EXAMPLE 4 Isolation of a cDNA Clone Encoding Der f VII from a λgt11 cDNALibrary

A λgt11 cDNA library was prepared from live adult Dermatophagoidesfarinae purchased from the Commonwealth Serum Laboratories, ParkvilleAustralia (Thomas, W. et al, Int. Arch Allergy Appl Immunol (1988)85:127-9). The library was prepared according to Trudinger et al.,(1991) Chem. Exp. Allergy, 21:33-37).

PCR amplification and DNA sequencing were used to isolate Der f VII cDNAfrom the λgt11 library. An oligonucleotide primer (Df1 in Table 1) basedon the predicted N-terminal sequence of Der p VII was made. This primerhad the sequence GCGAATTCGATCCAATTCACTATGAT-3′ (SEQ ID NO: 8). The firstGCGAATTC encodes an EcoRI site (GAATTC) and the sequence GAT encodes thefirst six residues of Der p VII. For the other primer, the λgt11GGTGGCGACGACTCCTGGAGCCCG-3′ (SEQ ID NO: 9) forward primer (Df2 inTable 1) flanking the EcoRI cloning site was used (New England Biolabs,Beverly, U.S.A.).

The PCR reactions were carried out in a final reaction volume of 50 μlcontaining 20 mM Tris-HC1 pH 8.2, 10 mM KC1, 6 mM (NH₄)₂SO₄, 2 mM MgCl₂,0.1% Triton X-100, 10 ngμ nuclease-free BSA, 10 mM dNTPs, 20 pmol ofeach primer and 2.5 units of Pfu DNA polymerase. This was obtained as akit from Stratagene (La Jolla, Calif., U.S.A.). Target DNA (λgt11 D.farinae cDNA ligations, 0.001 μg) was added and the contents of the tubewere mixed and overlayed with paraffin oil. The tubes were initiallydenatured at 95° C. for 5 min., then annealed at 55° C. for 2 min. andextended at 72° C. for 2 min. Thereafter for 48 cycles, denaturing wascarried out for 1 min. at 94° C. and annealing for 1 min. at 55° C. andextension as before. In the final (50th) cycle, the extension reactionwas increased to 10 min. to ensure that all amplified products were fulllength.

Ten microlitres of the reaction were then checked for amplified bands ona 1% agarose gel. The remainder of the reaction mixture was ethanolprecipitated prior to purification of the amplified product on a lowmelting point agarose gel (Bio-Rad., Richmond, U.S.A.).

The purified PCR product was digested with EcoRI and was ligated intothe M13 vector mp 18 (see Messing, supra), digested with EcoRi and thentransferred into E. coli strain TG1 competent cells. Isolated whiteplaques were picked and used to prepare phage stocks and single-strandedDNA for sequencing.

EXAMPLE 5 DNA Sequence Analysis of Der f VII cDNA

DNA sequencing was performed with the dideoxynucleotide chaintermination method using Sequenase version 2.0 (USB Corp., Cleveland,U.S.A.) according to the supplier's protocol. The primers used forsequencing include the M13 sequencing primer (−40) a 17-merGTTTTCCCAGTCACGAC-3′ (SEQ ID NO: 10) (Df3 in Table 1), the primer Df1(SEQ ID NO: 8) used for PCR reaction described in Example 4, and 2 otheroligonucleotide primers, Df4 and Df5, both shown in Table 1. The primerDf4 GGTGAATTAGCCATGCG-3′ (SEQ ID NO: 11) was previously used for thesequencing of Der p VII and primer Df5 TCAATCTTGGATCCAATTTTTGGC-3′ (SEQID NO: 12) was based on sequences of Der f VII from nucleotides 559-582.

To isolate a cDNA containing the 5′ untranslated region of Der f VII, anoligonucleotide primer based on the C terminal sequence of Der f VII wasmade. This primer (Df6 in Table 1) had the sequenceGGAATTCTTAATTTTTTTCCAATTCACG-3′ (SEQ ID NO: 13). The first GGAATTCencodes a EcoRI site. This sequence and the following sequence (TTA . .. ). are complementary to the reverse sequences of the stop codon andthe last six residues of Der f VII. For the other primer, the λgt11TTGACACCAGACCAACTGGTAATG-3′ reverse primer (SEQ ID NO: 14) (Df7 in Table1), flanking the EcoRI cloning site, was used.

The PCR reactions were carried out according to conditions described inExample 4. The PCR product was purified on a low melting point agarosegel, digested with EcoRI and was ligated into pUC 19 digested with EcoRIand then transferred into E. coli strain TGI competent cells. PlasmidDNA from transformant E. coli was isolated and used for sequencing.

DNA sequencing was performed using the same dideoxynucleotide chaintermination method, Sequence version 2.0, described above. However,before sequencing, the double-stranded plasmid DNA templates weredenatured by treatment with NaOH and neutralized by the addition ofsodium acetate and then ethanol precipitated according to the supplier'sprotocol. To obtain the 5′ untranslated end of Der f VII cDNA, theprimer Df8 (see Table 1) was used. This primer had the sequenceATGACGTTCGAATTTATC-3′ (SEQ ID NO: 15) which corresponds to the reversesequence of Der f VII from nucleotide no. 225-208.

TABLE 1 Oligonucleotides used for PCR amplication and sequencing derivedfrom Der f VII Name Sequence nucleotide positions DflGCGAATTCGATCCAATTCACTATGAT-3′ (SEQ ID NO:8) 94-111 Df2GGTGGCGACGACTCCTGGAGCCCG-3′ (SEQ ID NO:9) lambda gt11 forward primer Df3GTTTTCCCAGTCACGAC-3′ (SEQ ID NO:10) M13 sequencing primer (−40) Df4GGTGAATTAGACATGCG-3′ (SEQ ID NO:11) 247-263 Df5TCAATCTTGGATCCAATTTTTGGC-3′ (SEQ ID NO:12) 559-582 Df6CGAATTCTTAATTTTTTTCCAATTCACG-3′ (SEQ ID NO:13) 684-664 Df7TTGACACCAGACCAACTGGTAATG-3′ (SEQ ID NO:14) lambda gt11 reverse primerDf8 ATGACGTTCGAATTTATC-3′ (SEQ ID NO:15) 225-208

Equivalents

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, numerous equivalents to the specificembodiments described herein. Such equivalents are considered to bewithin the scope of this invention and are encompassed by the followingclaims.

15 812 base pairs nucleic acid single linear cDNA CDS 68..712mat_peptide 119..712 1 TTTTTTTTTT TTTTGGTTAT TCCCATTTTT TTCATATCGTAAAAATCCAA ATTCACTTTT 60 TTACCAA ATG ATG AAA TTA TTA TTG ATT GCT GCC GCAGCT TTT GTT GCC 109 Met Met Lys Leu Leu Leu Ile Ala Ala Ala Ala Phe ValAla -1 -5 -10 GTT TCG GCT GAT CCA ATT CAC TAT GAT AAA ATC ACC GAA GAAATT AAC 157 Val Ser Ala Asp Pro Ile His Tyr Asp Lys Ile Thr Glu Glu IleAsn -15 1 5 10 AAA GCT GTT GAT GAA GCC GTC GCT GCA ATT GAA AAA TCC GAAACA TTC 205 Lys Ala Val Asp Glu Ala Val Ala Ala Ile Glu Lys Ser Glu ThrPhe 15 20 25 GAT CCA ATG AAG GTA CCC GAT CAT TCT GAT AAA TTC GAA CGA CATATT 253 Asp Pro Met Lys Val Pro Asp His Ser Asp Lys Phe Glu Arg His Ile30 35 40 45 GGT ATC ATC GAT TTA AAA GGT GAA TTA GAC ATG CGA AAC ATT CAAGTT 301 Gly Ile Ile Asp Leu Lys Gly Glu Leu Asp Met Arg Asn Ile Gln Val50 55 60 CGA GGA TTA AAA CAA ATG AAA CGT GTA GGT GAT GCT AAT GTG AAA AGT349 Arg Gly Leu Lys Gln Met Lys Arg Val Gly Asp Ala Asn Val Lys Ser 6570 75 GAA GAT GGT GTT GTC AAA GCT CAT TTG TTG GTC GGT GTT CAT GAT GAC397 Glu Asp Gly Val Val Lys Ala His Leu Leu Val Gly Val His Asp Asp 8085 90 GTT GTT TCA ATG GAA TAT GAT TTA GCA TAC AAA TTG GGT GAT CTT CAT445 Val Val Ser Met Glu Tyr Asp Leu Ala Tyr Lys Leu Gly Asp Leu His 95100 105 CCA AAC ACT CAT GTC ATT TCG GAT ATT CAG GAT TTT GTT GTC GAA TTA493 Pro Asn Thr His Val Ile Ser Asp Ile Gln Asp Phe Val Val Glu Leu 110115 120 125 TCG CTC GAA GTT AGC GAA GAA GGT AAT ATG ACA TTG ACA TCG TTCGAA 541 Ser Leu Glu Val Ser Glu Glu Gly Asn Met Thr Leu Thr Ser Phe Glu130 135 140 GTA CGT CAA TTT GCC AAT GTT GTC AAT CAT ATT GGT GGT CTT TCAATT 589 Val Arg Gln Phe Ala Asn Val Val Asn His Ile Gly Gly Leu Ser Ile145 150 155 TTG GAT CCA ATT TTC GCT GTC TTA TCC GAT GTT TTG ACC GCT ATTTTC 637 Leu Asp Pro Ile Phe Ala Val Leu Ser Asp Val Leu Thr Ala Ile Phe160 165 170 CAG GAT ACC GTA CGT GCA GAA ATG ACC AAA GTA TTG GCA CCA GCATTC 685 Gln Asp Thr Val Arg Ala Glu Met Thr Lys Val Leu Ala Pro Ala Phe175 180 185 AAA AAA GAA TTG GAA CGA AAC AAC CAA TAGACTTACA CACAACATAA732 Lys Lys Glu Leu Glu Arg Asn Asn Gln 190 195 CACTGTTATT TTTACACTGGATAATCAAAT GAAATAAATT TTTTTATCAT TTTGTTTAAA 792 AAAAAAAAAA AAAAAAAAAA812 215 amino acids amino acid linear protein 2 Met Met Lys Leu Leu LeuIle Ala Ala Ala Ala Phe Val Ala Val Ser -1 -5 -10 -15 Ala Asp Pro IleHis Tyr Asp Lys Ile Thr Glu Glu Ile Asn Lys Ala 1 5 10 15 Val Asp GluAla Val Ala Ala Ile Glu Lys Ser Glu Thr Phe Asp Pro 20 25 30 Met Lys ValPro Asp His Ser Asp Lys Phe Glu Arg His Ile Gly Ile 35 40 45 Ile Asp LeuLys Gly Glu Leu Asp Met Arg Asn Ile Gln Val Arg Gly 50 55 60 Leu Lys GlnMet Lys Arg Val Gly Asp Ala Asn Val Lys Ser Glu Asp 65 70 75 Gly Val ValLys Ala His Leu Leu Val Gly Val His Asp Asp Val Val 80 85 90 95 Ser MetGlu Tyr Asp Leu Ala Tyr Lys Leu Gly Asp Leu His Pro Asn 100 105 110 ThrHis Val Ile Ser Asp Ile Gln Asp Phe Val Val Glu Leu Ser Leu 115 120 125Glu Val Ser Glu Glu Gly Asn Met Thr Leu Thr Ser Phe Glu Val Arg 130 135140 Gln Phe Ala Asn Val Val Asn His Ile Gly Gly Leu Ser Ile Leu Asp 145150 155 Pro Ile Phe Ala Val Leu Ser Asp Val Leu Thr Ala Ile Phe Gln Asp160 165 170 175 Thr Val Arg Ala Glu Met Thr Lys Val Leu Ala Pro Ala PheLys Lys 180 185 190 Glu Leu Glu Arg Asn Asn Gln 195 18 base pairsnucleic acid single linear cDNA 3 GATCCAATTC ACTATGAT 18 17 base pairsnucleic acid single linear cDNA 4 GGTGAATTAG ACATGCG 17 24 base pairsnucleic acid single linear cDNA 5 TCAATTTTGG ATCCAATTTT CGCT 24 761 basepairs nucleic acid single linear cDNA CDS 43..681 6 GATCTTATATCAATAACAAT CCAAAAAAAC ATATCTTACA AA ATG ATG AAA TTT 54 Met Met Lys Phe 1TTG TTG ATT GCT GCC GTG GCA TTT GTC GCC GTT TCG GCT GAT CCA ATT 102 LeuLeu Ile Ala Ala Val Ala Phe Val Ala Val Ser Ala Asp Pro Ile 5 10 15 20CAC TAT GAT AAA ATC ACC GAA GAA ATC AAC AAA GCT ATT GAT GAT GCC 150 HisTyr Asp Lys Ile Thr Glu Glu Ile Asn Lys Ala Ile Asp Asp Ala 25 30 35 ATTGCT GCT ATT GAA CAA TCC GAA ACA ATA GAT CCA ATG AAA GTA CCT 198 Ile AlaAla Ile Glu Gln Ser Glu Thr Ile Asp Pro Met Lys Val Pro 40 45 50 GAT CATGCC GAT AAA TTC GAA CGT CAT GTT GGT ATT GTG GAT TTC AAA 246 Asp His AlaAsp Lys Phe Glu Arg His Val Gly Ile Val Asp Phe Lys 55 60 65 GGT GAA TTAGCC ATG CGA AAC ATT GAG GCT CGA GGA TTG AAA CAA ATG 294 Gly Glu Leu AlaMet Arg Asn Ile Glu Ala Arg Gly Leu Lys Gln Met 70 75 80 AAA CGT CAA GGTGAT GCT AAT GTC AAA GGT GAA GAG GGT ATT GTT AAA 342 Lys Arg Gln Gly AspAla Asn Val Lys Gly Glu Glu Gly Ile Val Lys 85 90 95 100 GCT CAT TTG TTGATC GGT GTT CAC GAT GAT ATC GTC TCG ATG GAA TAT 390 Ala His Leu Leu IleGly Val His Asp Asp Ile Val Ser Met Glu Tyr 105 110 115 GAT TTA GCA TACAAA TTG GGT GAT CTT CAT CCA ACC ACT CAT GTC ATT 438 Asp Leu Ala Tyr LysLeu Gly Asp Leu His Pro Thr Thr His Val Ile 120 125 130 TCG GAT ATT CAAGAT TTT GTT GTT GCC TTG TCC CTT GAA ATT TCT GAT 486 Ser Asp Ile Gln AspPhe Val Val Ala Leu Ser Leu Glu Ile Ser Asp 135 140 145 GAA GGT AAC ATAACA ATG ACA TCT TTT GAA GTA CGA CAA TTC GCT AAT 534 Glu Gly Asn Ile ThrMet Thr Ser Phe Glu Val Arg Gln Phe Ala Asn 150 155 160 GTT GTC AAC CATATT GGT GGT CTT TCA ATC TTG GAT CCA ATT TTT GGC 582 Val Val Asn His IleGly Gly Leu Ser Ile Leu Asp Pro Ile Phe Gly 165 170 175 180 GTT TTA TCTGAT GTA TTG ACC GCT ATT TTC CAA GAC ACC GTA CGT AAG 630 Val Leu Ser AspVal Leu Thr Ala Ile Phe Gln Asp Thr Val Arg Lys 185 190 195 GAA ATG ACCAAA GTA TTG GCA CCA GCA TTT AAA CGT GAA TTG GAA AAA 678 Glu Met Thr LysVal Leu Ala Pro Ala Phe Lys Arg Glu Leu Glu Lys 200 205 210 AATTAACCAATAG ACATCATTTT TCCAACTGTA CAATCTCTAT TTCACTGACA 731 AsnATAAAATAAA ATTTTTATTT TTATTTCTCC 761 213 amino acids amino acid linearprotein 7 Met Met Lys Phe Leu Leu Ile Ala Ala Val Ala Phe Val Ala ValSer 1 5 10 15 Ala Asp Pro Ile His Tyr Asp Lys Ile Thr Glu Glu Ile AsnLys Ala 20 25 30 Ile Asp Asp Ala Ile Ala Ala Ile Glu Gln Ser Glu Thr IleAsp Pro 35 40 45 Met Lys Val Pro Asp His Ala Asp Lys Phe Glu Arg His ValGly Ile 50 55 60 Val Asp Phe Lys Gly Glu Leu Ala Met Arg Asn Ile Glu AlaArg Gly 65 70 75 80 Leu Lys Gln Met Lys Arg Gln Gly Asp Ala Asn Val LysGly Glu Glu 85 90 95 Gly Ile Val Lys Ala His Leu Leu Ile Gly Val His AspAsp Ile Val 100 105 110 Ser Met Glu Tyr Asp Leu Ala Tyr Lys Leu Gly AspLeu His Pro Thr 115 120 125 Thr His Val Ile Ser Asp Ile Gln Asp Phe ValVal Ala Leu Ser Leu 130 135 140 Glu Ile Ser Asp Glu Gly Asn Ile Thr MetThr Ser Phe Glu Val Arg 145 150 155 160 Gln Phe Ala Asn Val Val Asn HisIle Gly Gly Leu Ser Ile Leu Asp 165 170 175 Pro Ile Phe Gly Val Leu SerAsp Val Leu Thr Ala Ile Phe Gln Asp 180 185 190 Thr Val Arg Lys Glu MetThr Lys Val Leu Ala Pro Ala Phe Lys Arg 195 200 205 Glu Leu Glu Lys Asn210 26 base pairs nucleic acid single linear cDNA 8 GCGAATTCGATCCAATTCAC TATGAT 26 24 base pairs nucleic acid single linear cDNA 9GGTGGCGACGACTCCTGGAGCCCG 24 17 base pairs nucleic acid single linearcDNA 10 GTTTTCCCAGTCACGAC 17 17 base pairs nucleic acid single linearcDNA 11 GGTGAATTAGACATGCG 17 24 base pairs nucleic acid single linearcDNA 12 TCAATCTTGGATCCAATTTTTGGC 24 28 base pairs nucleic acid singlelinear cDNA 13 GGAATTCTTA ATTTTTTTCC AATTCACG 28 24 base pairs nucleicacid single linear cDNA 14 TTGACACCAGACCAACTGGTAATG 24 18 base pairsnucleic acid single linear cDNA 15 ATGACGTTCGAATTTATC 18

What is claimed is:
 1. An isolated Group VII protein allergen ofDermatophagoides farinae, Der f VII, comprising the amino acid sequenceshown in FIGS. 6A and 6B (SEQ ID NO:7) or the mature portion thereof. 2.A composition comprising the Der f VII protein allergen of claim 1 and apharmaceutically acceptable carrier.
 3. An isolated Group VII proteinallergen of Dermatophagoides farinae, Der f VII, encoded by the nucleicacid shown in FIGS. 6A and 6B (SEQ ID NO: 6).
 4. An isolated Group VIIprotein allergen of Dermatophagoides farinae, Der f VII, encoded by thenucleotide bases 43 through 681 of the nucleic acid shown in FIG. 6A and6B (SEQ ID NO: 6).
 5. A composition comprising the Der f VII proteinallergen of either claim 3 or claim 4 and a pharmaceutically acceptablecarrier.
 6. The isolated peptide of claim 1, produced by chemicalsynthesis.
 7. The isolated peptide of claim 1, produced by recombinantexpression.
 8. A method of producing an isolated Group VII proteinallergen of Dermatophagoides farinae, Der f VII, or the mature portionthereof, the method being selected from the group consisting of: a)recombinantly producing the protein a host cell transformed with anucleic acid encoding a Der f VII protein allergen comprising the aminoacid sequence shown in FIGS. 6A and 6B (SEQ ID NO:7), or the matureportion thereof; and b) chemically synthesizing the protein, wherein theprotein comprises the amino acid sequence shown in FIGS. 6A and 6B (SEQID No: 7) or the mature protein thereof.
 9. The method of claim 8wherein the nucleic acid comprises the nucleotide sequence shown inFIGS. 6A and 6B (SEQ ID NO: 6).